Acupuncture treatment is based on acupoint stimulation; however, the biological basis is not understood. We stimulated one acupoint with catgut embedding (CEP) for 8 weeks and then used isobaric tags for relative and absolute quantitation to screen proteins with altered expression in adjacent acupoints of Sprague Dawley rats. The expression levels of 10 proteins, including kininogen (KNG), consistently changed >1.5-fold in all three acupoints in CEP versus sham CEP-treated rats. Eight-week, but not 3-day, CEP treatment enhanced KNG expression. The enhanced KNG expression among small vessels in the subcutaneous layer was revealed via immunofluorescence. Nitric oxide synthesis was enhanced similar to KNG. These findings uncover biological changes at acupoints and suggest the critical role of the KNG–nitric oxide signaling pathway in acupoint activation.